Spiro heterocycles bearing piperidine moiety as potential scaffold for antileishmanial activity: synthesis, biological evaluation, and in silico studies.

New spiro-piperidine derivatives were synthesised via the eco-friendly ionic liquids in a one-pot fashion. The in vitro antileishmanial activity against Leishmania major promastigote and amastigote forms highlighted promising antileishmanial activity for most of the derivatives, with superior activity compared to miltefosine. The most active compounds 8a and 9a exhibited sub-micromolar range of activity, with IC50 values of 0.89 µM and 0.50 µM, respectively, compared to 8.08 µM of miltefosine. Furthermore, the antileishmanial activity reversal of these compounds via folic and folinic acids displayed comparable results to the positive control trimethoprim. This emphasises that their antileishmanial activity is through the antifolate mechanism via targeting DHFR and PTR1. The most active compounds showed superior selectivity and safety profile compared to miltefosine against VERO cells. Moreover, the docking experiments of 8a and 9a against Lm-PTR1 rationalised the observed in vitro activities. Molecular dynamics simulations confirmed a stable and high potential binding to Lm-PTR1.

New pyrazolylpyrazoline derivatives as dual acting antimalarial-antileishamanial agents: synthesis, biological evaluation and molecular modelling simulations.

Promising inhibitory activities of the parasite multiplication were obtained upon evaluation of in vivo antimalarial activities of new pyrazolylpyrazoline derivatives against Plasmodium berghei infected mice. Further evaluation of 5b and 6a against chloroquine-resistant strain (RKL9) of P. falciparum showed higher potency than chloroquine. In vitro antileishmanial activity testing against Leishmania aethiopica promastigote and amastigote forms indicated that 5b, 6a and 7b possessed promising activity compared to miltefosine and amphotericin B deoxycholate. Moreover, antileishmanial activity reversal of the active compounds via folic and folinic acids showed comparable results to the positive control trimethoprim, indicating an antifolate mechanism via targeting leishmanial DHFR and PTR1. The compounds were non-toxic at 125, 250 and 500 mg/kg. In addition, docking of the most active compound against putative malarial target Pf-DHFR-TS and leishmanial PTR1 rationalised the observed activities. Molecular dynamics simulations confirmed a stable and high potential binding of 7a against leishmanial PTR1.

Protective Effect of TRPM8 against Indomethacin-Induced Small Intestinal Injury via the Release of Calcitonin Gene-Related Peptide in Mice.

Transient receptor potential melastatin 8 (TRPM8) is a non-selective cation channel activated by mild cooling and chemical agents including menthol. Nonsteroidal anti-inflammatory drugs have antipyretic, analgesic effects, and they can cause stomach and small intestinal injury. The current study investigated the role of TRPM8 in the pathogenesis of indomethacin-induced small intestinal injury. In male TRPM8-deficient (TRPM8KO) and wild-type (WT) mice, intestinal injury was induced via the subcutaneous administration of indomethacin. In addition, the effect of WS-12, a specific TRPM8 agonist, was examined in TRPM8KO and WT mice with indomethacin-induced intestinal injury. TRPM8KO mice had a significantly higher intestinal ulcerogenic response to indomethacin than WT mice. The repeated administration of WS-12 significantly attenuated the severity of intestinal injury in WT mice. However, this response was abrogated in TRPM8KO mice. Furthermore, in TRPM8-enhanced green fluorescent protein (EGFP) transgenic mice, which express EGFP under the direction of TRPM8 promoter, the EGFP signals in the indomethacin-treated intestinal mucosa were upregulated. Further, the EGFP signals were commonly found in calcitonin gene-related peptide (CGRP)-positive sensory afferent neurons and partly colocalized with substance P (SP)-positive neurons in the small intestine. The intestinal CGRP-positive neurons were significantly upregulated after the administration of indomethacin in WT mice. Nevertheless, this response was abrogated in TRPM8KO mice. In contrast, indomethacin increased the expression of intestinal SP-positive neurons in not only WT mice but also TRPM8KO mice. Thus, TRPM8 has a protective effect against indomethacin-induced small intestinal injury. This response may be mediated by the upregulation of CGRP, rather than SP.

Leukotriene B4 Receptor Type 2 Accelerates the Healing of Intestinal Lesions by Promoting Epithelial Cell Proliferation.

Leukotriene B4 receptor type 2 (BLT2) is a low-affinity leukotriene B4 receptor that is highly expressed in intestinal epithelial cells. Previous studies demonstrated the protective role of BLT2 in experimentally induced colitis. However, its role in intestinal lesion repair is not fully understood. We investigated the role of BLT2 in the healing of indomethacin-induced intestinal lesions in mice. There was no significant different between wild-type (WT) and BLT2-deficient (BLT2KO) mice in terms of the development of indomethacin-induced intestinal lesions. However, healing of these lesions was significantly impaired in BLT2KO mice compared with WT mice. In contrast, transgenic mice with intestinal epithelium-specific BLT2 overexpression presented with superior ileal lesion healing relative to WT mice. An immunohistochemical study showed that the number of Ki-67-proliferative cells was markedly increased during the healing of intestinal lesions in WT mice but significantly attenuated in BLT2KO mice. Exposure of cultured mouse intestinal epithelial cells to CAY10583, a BLT2 agonist, promoted wound healing and cell proliferation in a concentration-dependent manner. Nevertheless, these responses were abolished under serum-free conditions. The CAY10583-induced proliferative effect was also negated by Go6983, a protein kinase C (PKC) inhibitor, U-73122, a phospholipase C (PLC) inhibitor, LY255283, a BLT2 antagonist, and pertussis toxin that inhibits G protein-coupled receptor signaling via Gi/o proteins. Thus, BLT2 plays an important role in intestinal wound repair. Moreover, this effect is mediated by the promotion of epithelial cell proliferation via the Gi/o protein-dependent and PLC/PKC signaling pathways. The BLT2 agonists are potential therapeutic agents for the treatment of intestinal lesions. SIGNIFICANCE STATEMENT: The healing of indomethacin-induced Crohn's disease-like intestinal lesions was impaired in mice deficient in low-affinity leukotriene B4 receptor type 2 (BLT2). They presented with reduced epithelial cell proliferation during the healing. In contrast, healing was promoted in mice overexpressing intestinal epithelial BLT2. In cultured intestinal epithelial cells, the BLT2 agonist CAY10583 substantially accelerated wound repair by enhancing cell proliferation rather than migration. Thus, BLT2 plays an important role in the intestinal lesions via acceleration of epithelial cell proliferation.

Role of transient receptor potential melastatin 2 in surgical inflammation and dysmotility in a mouse model of postoperative ileus.

In this study, we investigated the role of transient receptor potential melastatin 2 (TRPM2), a nonselective cation channel abundantly expressed in inflammatory cells such as macrophages, in the development of postoperative ileus, a complication of abdominal surgery characterized by gastrointestinal dysmotility. In wild-type mice, we found that intestinal manipulation, a maneuver that elicits symptoms typical of postoperative ileus, delays the transit of fluorescein-labeled dextran, promotes the infiltration of CD68+ macrophages, Ly6B.2+ neutrophils, and MPO+ cells into intestinal muscles, boosts expression of IL-1ß, IL-6, TNF-α, iNOS, and CXCL2 in intestinal muscles and peritoneal macrophages, enhances phosphorylation of ERK and p38 MAPK in intestinal muscles, and amplifies IL-1ß, IL-6, TNF-α, iNOS, and CXCL2 expression in resident and thioglycolate-elicited peritoneal macrophages following exposure to lipopolysaccharide. Remarkably, TRPM2 deficiency completely blocks or diminishes these effects. Indeed, intestinal manipulation appears to activate TRPM2 in resident muscularis macrophages and elicits release of inflammatory cytokines and chemokines, which, in turn, promote infiltration of macrophages and neutrophils into the muscle, ultimately resulting in dysmotility. NEW & NOTEWORTHY Activation of transient receptor potential melastatin 2 (TRPM2) releases inflammatory cytokines and chemokines, which, in turn, promote the infiltration of inflammatory cells and macrophages into intestinal muscles, ultimately resulting in dysmotility. Thus TRPM2 is a promising target in treating dysmotility due to postoperative ileus, a complication of abdominal surgery.

Transient receptor potential vanilloid 1 and transient receptor potential ankyrin 1 contribute to the progression of colonic inflammation in dextran sulfate sodium-induced colitis in mice: Links to calcitonin gene-related peptide and substance P.

Transient receptor potential (TRP) vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1), which are non-selective cation channels, play important roles in the sensation of pain. This study investigated the roles of TRPV1 and TRPA1 in dextran sulfate sodium (DSS)-induced murine colitis. DSS (2%) administered for 7 days caused severe colitis that was significantly less severe in TRPV1-deficient (TRPV1KO) and TRPA1-deficient (TRPA1KO) mice than that in wild-type (WT) mice. Similar colitis attenuations were observed in TRPV1KO and TRPA1KO mice but not in WT mice that had been transplanted with bone marrow cells from WT, TRPA1KO, or TRPV1KO mice. DSS treatment upregulated calcitonin gene-relative peptide (CGRP)- and substance P (SP)-positive nerve fibers in the colonic mucosa of WT mice. TRPV1KO and TRPA1KO mice showed significant reductions in the DSS-induced upregulation of SP, but the DSS-induced upregulation of CGRP was not reduced. Sensory deafferentation evoked by pretreatment with high doses of capsaicin markedly exacerbated DSS-induced colitis with reductions in DSS-induced upregulation of SP- and CGRP-positive nerve fibers. These findings suggest that neuronal TRPV1 and TRPA1 contribute to the progression of colonic inflammation. While these responses may be mediated by the upregulation of SP-mediated deleterious mechanisms, CGRP may be associated with protective mechanisms.

NOX1/NADPH Oxidase Expressed in Colonic Macrophages Contributes to the Pathogenesis of Colonic Inflammation in Trinitrobenzene Sulfonic Acid-Induced Murine Colitis.

NOX1/NADPH oxidase, a nonphagocytic isoform of reactive oxygen species-producing enzymes, is highly expressed in the colon, but the physiologic and pathophysiologic roles of this isoform are not fully understood. The present study investigated the role of NOX1 in the development of colonic inflammation in a trinitrobenzene sulfonic acid (TNBS)-induced murine colitis model. Intrarectal injection of TNBS caused severe colitis accompanied by body weight loss, diarrhea, and increased myeloperoxidase (MPO) activity in wild-type (WT) mice. In contrast, the severity of colitis was significantly attenuated in NOX1-deficient (NOX1KO) mice (the inhibitions of macroscopic damage score, body weight loss, diarrhea score, and MPO activity were 73.1%, 36.8%, 83.3%, and 98.4%, respectively). TNBS-induced upregulation of inflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-1ß), chemokines (CXCL1 and CXLC2), and inducible nitric oxide synthase (iNOS) was also significantly less in NOX1KO than in WT mice (the inhibitions were 100.8%, 89.0%, 63.5%, 96.7%, and 97.1%, respectively). Expression of NOX1 mRNA was detected not only in the lamina propria but also in peritoneal macrophages isolated from WT mice. Increased expression of TNF-α, IL-1ß, and iNOS in peritoneal macrophages exposed to lipopolysaccharide was significantly attenuated in macrophages isolated from NOX1KO mice (68.1%, 67.0%, and 79.3% inhibition, respectively). These findings suggest that NOX1/NADPH oxidase plays an important role in the pathogenesis of TNBS-induced colonic inflammation via upregulation of inflammatory cytokines, chemokines, and iNOS. NOX1 in colonic macrophages may become a potential target in pharmacologic intervention for inflammatory bowel disease.

G protein-coupled receptor 35 contributes to mucosal repair in mice via migration of colonic epithelial cells.

G protein-coupled receptor 35 (GPR35), a receptor for lysophosphatidic acid, is highly expressed in the gastrointestinal tract. Recently, GPR35 has been implicated in the onset of inflammatory bowel disease (IBD), but its role in physiological and pathological processes in the colon remains undefined. In this study, we investigated the contribution of GPR35-mediated signalling to mucosal repair of colonic epithelium in IBD. GPR35 function was examined in a wound healing model, using young adult mouse colon epithelium (YAMC) cells, and in a dextran sulphate sodium (DSS)-induced mouse model of colitis. Cell proliferation, mRNA expression, extracellular signal-regulated kinase (ERK) activation, and protein localization were determined by MTT assay, quantitative RT-PCR, western blotting, and immunohistochemistry, respectively. GPR35 agonists (YE120, zaprinast, and pamoic acid) promoted wound repair in a concentration-dependent manner independently of cell proliferation, whereas a specific GPR35 antagonist CID2745687, forskolin, and pertussis toxin reversed the YE120-induced effect. YE120 increased the mRNA expression of fibronectin and its receptor integrin α5, and ERK1/2 phosphorylation, but these responses were attenuated by CID2745687 and forskolin. Furthermore, the severity of DSS-induced colitis was significantly reduced by daily injections of pamoic acid via upregulation of fibronectin and integrin α5 in the colonic epithelium. GPR35 signalling promotes mucosal repair by inducing fibronectin and integrin α5 expression, coupling to Gi protein, and activating ERK1/2 in colonic epithelial cells. These findings define GPR35 as a candidate therapeutic target in IBD.

Probiotic Bifidobacterium bifidum G9-1 attenuates 5-fluorouracil-induced intestinal mucositis in mice via suppression of dysbiosis-related secondary inflammatory responses.

Bifidobacterium, a major component of the intestinal microbiota, has been clinically used for the treatment of diarrhoea and constipation. 5-Fluorouracil (5-FU), widely used for cancer chemotherapy, is known to frequently induce intestinal mucositis accompanied by severe diarrhoea. The present study examined the effect of Bifidobacterium bifidum G9-1 (BBG9-1) on 5-FU-induced intestinal mucositis in mice. Intestinal mucositis was induced by repeated administration of 5-FU for 6 days. BBG9-1 was administered orally once daily for 9 days, beginning 3 days before the onset of 5-FU treatment. Repeated administration of 5-FU caused severe intestinal mucositis, characterised by shortening of villi and destruction of crypts, accompanied by increases in intestinal myeloperoxidase activity and inflammatory cytokine expression, body weight loss, and diarrhoea on day 6. Daily administration of BBG9-1 significantly reduced the severity of intestinal mucositis and inflammatory responses and tended to attenuate clinical symptoms. In contrast, BBG9-1 failed to prevent apoptosis induction on day 1 after the first 5-FU administration. The structure of the intestinal microbiota, as analysed by weighted UniFrac distance, was largely altered by 5-FU treatment, but this change was mitigated by daily administration of BBG9-1. Moreover, 5-FU treatment decreased the abundance of Firmicutes and increased the abundance of Bacteroidetes, but these responses were also significantly inhibited by daily administration of BBG9-1. These results suggest that BBG9-1 has an ameliorative effect against 5-FU-induced intestinal mucositis through the attenuation of inflammatory responses via improve dysbiosis. BBG9-1 could be useful for the prevention of intestinal mucositis during cancer chemotherapy.

Mucosal protective agents prevent exacerbation of NSAID-induced small intestinal lesions caused by antisecretory drugs in rats.

Antisecretory drugs such as histamine H2-receptor antagonists and proton pump inhibitors are commonly used for the treatment of upper gastrointestinal mucosal lesions induced by nonsteroidal anti-inflammatory drugs (NSAIDs). However, it has recently been reported that these drugs exacerbate NSAID-induced small intestinal lesions in rats. Unfortunately, there are few effective agents for the treatment of this complication. We examined the effects of mucosal protective agents (MPAs) (misoprostol, irsogladine, and rebamipide) and mucin of porcine stomach on diclofenac-induced intestinal lesions and the exacerbation of the lesions by ranitidine or omeprazole. The effects of the drugs on intestinal motility and mucus distribution/content were also examined. Male Wistar rats (180-220 g) were used. Each drug was administered orally under fed conditions. Diclofenac (1-10 mg/kg) produced multiple lesions in the small intestine dose-dependently. Both ranitidine (30 mg/kg) and omeprazole (100 mg/kg) significantly increased the intestinal lesions induced by low doses (3 and 6 mg/kg) of diclofenac. Misoprostol (0.03-0.3 mg/kg), irsogladine (3-30 mg/kg), and rebamipide (30-300 mg/kg), as well as mucin (30-300 mg/kg) inhibited the formation of intestinal lesions caused by a high dose (10 mg/kg) of diclofenac alone and prevented the exacerbation of diclofenac-induced lesions by antisecretory drugs. Diclofenac (10 mg/kg) markedly increased the intestinal motility and decreased the mucosal mucus, and the decrease of mucus was significantly inhibited by the MPAs. These results indicate the usefulness of the MPAs for the treatment of intestinal lesions induced by NSAIDs alone or by coadministration with antisecretory drugs, and suggest that mucus plays an important role in the protection of intestinal mucosa by the MPAs.

Lubiprostone prevents nonsteroidal anti-inflammatory drug-induced small intestinal damage by suppressing the expression of inflammatory mediators via EP4 receptors.

Lubiprostone, a bicyclic fatty acid derived from prostaglandin E1, has been used to treat chronic constipation and irritable bowel syndrome, and its mechanism of action has been attributed to the stimulation of intestinal fluid secretion via the activation of the chloride channel protein 2/cystic fibrosis transmembrane regulator (ClC-2/CFTR) chloride channels. We examined the effects of lubiprostone on indomethacin-induced enteropathy and investigated the functional mechanisms involved, including its relationship with the EP4 receptor subtype. Male Sprague-Dawley rats were administered indomethacin (10 mg/kg p.o.) and killed 24 hours later to examine the hemorrhagic lesions that developed in the small intestine. Lubiprostone (0.01-1 mg/kg) was administered orally twice 30 minutes before and 9 h after the indomethacin treatment. Indomethacin markedly damaged the small intestine, accompanied by intestinal hypermotility, a decrease in mucus and fluid secretion, and an increase in enterobacterial invasion as well as the up-regulation of inducible nitric-oxide synthase (iNOS) and tumor necrosis factor α (TNFα) mRNAs. Lubiprostone significantly reduced the severity of these lesions, with the concomitant suppression of the functional changes. The effects of lubiprostone on the intestinal lesions and functional alterations were significantly abrogated by the coadministration of AE3-208 [4-(4-cyano-2-(2-(4-fluoronaphthalen-1-yl)propionylamino)phenyl)butyric acid], a selective EP4 antagonist, but not by CFTR(inh)-172, a CFTR inhibitor. These results suggest that lubiprostone may prevent indomethacin-induced enteropathy via an EP4 receptor-dependent mechanism. This effect may be functionally associated with the inhibition of intestinal hypermotility and increase in mucus/fluid secretion, resulting in the suppression of bacterial invasion and iNOS/TNFα expression, which are major pathogenic events in enteropathy. The direct activation of CFTR/ClC-2 chloride channels is not likely to have contributed to the protective effects of lubiprostone.

Distribution of transient receptor potential cation channel subfamily V member 1-expressing nerve fibers in mouse esophagus.

Transient receptor potential cation channel subfamily V member 1 (TRPV1) plays a role in esophageal function. However, the distribution of TRPV1 nerve fibers in the esophagus is currently not well understood. In the present study, we investigated the distribution of TRPV1 and neurotransmitters released from TRPV1 nerve fibers in the mouse lower esophagus. Furthermore, we investigated changes in the presence of TRPV1 in the mouse model of esophagitis. Numerous TRPV1-immunoreactive nerve fibers were seen in both the submucosal layer and myenteric plexus of the lower esophagus and colocalized with calcitonin gene-related peptide (CGRP). TRPV1 colocalized with substance P in axons in the submucosal layer and myenteric plexus. TRPV1 colocalized with neuronal nitric oxide synthase in the myenteric plexus. We observed some colocalization of CGRP with the vesicular acetylcholine (ACh) transporter, packaging of ACh into synaptic vesicles after its synthesis in terminal cytoplasm, in the submucosal layer and myenteric plexus. In the esophagitis model, the number of the TRPV1 nerve fibers did not change, but their immunoreactive intensity increased compared with sham-operated mice. Inhibitory effect of exogenous capsaicin on electrically stimulated twitch contraction significantly increased in esophagitis model compared with the effect in sham-operated mice. Overall, these results suggest that TRPV1 nerve fibers projecting to both the submucosal and muscle layer of the esophagus are extrinsic spinal and vagal afferent neurons. Furthermore, TRPV1 nerve fibers contain CGRP, substance P, nitric oxide, and ACh. Therefore, acid influx-mediated TRPV1 activation may play a role in regulating esophageal relaxation.

Importance of cyclooxygenase-1/prostacyclin in modulating gastric mucosal integrity under stress conditions.

BACKGROUND AND AIM: We investigated the roles of cyclooxygenase (COX) isozymes and prostaglandins (PGs) and their receptors in mucosal defense against cold-restraint stress (CRS)-induced gastric lesions. METHODS: Male C57BL/6 wild-type (WT) mice and those lacking COX-1 or COX-2 as well as those lacking EP1, EP3, or IP receptors were used after 18 h fasting. Animals were restrained in Bollman cages and kept in a cold room at 10°C for 90 min. RESULTS: CRS induced multiple hemorrhagic lesions in WT mouse stomachs. The severity of these lesions was significantly worsened by pretreatment with the nonselective COX inhibitors (indomethacin, loxoprofen) or selective COX-1 inhibitor (SC-560), while neither of the selective COX-2 inhibitors (rofecoxib and celecoxib) had any effect. These lesions were also aggravated in animals lacking COX-1, but not COX-2. The expression of COX-2 mRNA was not detected in the stomach after CRS, while COX-1 expression was observed under normal and stressed conditions. The gastric ulcerogenic response to CRS was similar between EP1 or EP3 knockout mice and WT mice, but was markedly worsened in animals lacking IP receptors. Pretreating WT mice with iloprost (the PGI2 analog) significantly prevented CRS-induced gastric lesions in the presence of indomethacin. PGE2 also reduced the severity of these lesions, and the effect was mimicked by the EP4 agonist, AE1-329. CONCLUSIONS: These results suggest that endogenous PGs derived from COX-1 play a crucial role in gastric mucosal defense during CRS, and this action is mainly mediated by PGI2 /IP receptors and partly by PGE2 /EP4 receptors.

Effect of rebamipide on gastric bleeding and ulcerogenic responses induced by aspirin plus clopidogrel under stimulation of acid secretion in rats.

BACKGROUND AND AIM: We examined the prophylactic effect of rebamipide on gastric bleeding induced by the perfusion of aspirin (acetylsalicylic acid [ASA]) plus clopidogrel under the stimulation of acid secretion in rats. METHODS: Under urethane anesthesia, acid secretion was stimulated by the i.v. infusion of histamine (8 mg/kg/h), and the stomach was perfused with 25 mmol/L ASA at a rate of 0.4 mL/min. Gastric bleeding was evaluated as the concentration of hemoglobin in the perfusate. Clopidogrel (30 mg/kg) was given p.o. 24 h before the perfusion. Rebamipide (3-30 mg/kg) or other antiulcer drugs were given i.d. before the ASA perfusion. RESULTS: Slight gastric bleeding or damage was observed with the perfusion of ASA under the stimulation of acid secretion, whereas these responses were significantly increased in the presence of clopidogrel. Both omeprazole and famotidine inhibited acid secretion and prevented these responses to ASA plus clopidogrel. Rebamipide had no effect on acid secretion, but dose-dependently prevented gastric bleeding in response to ASA plus clopidogrel, with the degree of inhibition being almost equivalent to that of the antisecretory drugs, and the same effects were obtained with the gastroprotective drugs, irsogladine and teprenone. These agents also reduced the severity of gastric lesions, although the effects were less than those of the antisecretory drugs. CONCLUSIONS: These results suggest that the antiplatelet drug, clopidogrel, increases gastric bleeding induced by ASA under the stimulation of acid secretion, and the gastroprotective drug, rebamipide, is effective in preventing the gastric bleeding induced under such conditions, similar to antisecretory drugs.

Orally Administrated Glutamate Restored EAAT1 and 3 Expression Levels Suppressed in 5-Fluorouracil-damaged Intestinal Epithelial Cells.

BACKGROUND/AIM: 5-Fluorouracil (5-FU) treatment induces intestinal mucositis, with diarrhea as the primary symptom. Mucositis significantly reduces patients' quality of life (QOL). Amino acids such as glutamate are beneficial for treating gastrointestinal disorders; however, the underlying mechanism remains unclear. Therefore, this study aimed to clarify the role of excitatory amino acid transporters (EAATs) in 5-FU-induced intestinal injury. MATERIALS AND METHODS: The rat intestinal epithelial cell line (IEC-6) was used to evaluate whether the EAAT inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC) affects 5-FU-induced cytotoxicity. Mice with 5-FU-induced mucositis were used to determine the effects of glutamate on EAATs expression levels. RESULTS: Treatment with L-trans-PDC suppressed IEC-6 cell growth. It also exacerbated the 5-FU-induced cell growth suppression and increased inflammatory cytokine expression. In addition, mice treated with 5-FU+Glutamate showed higher EAAT1,3 expression than 5-FU only-treated mice. CONCLUSION: Decreased EAAT levels worsen intestinal cell damage caused by 5-FU, suppress cell growth, and induce inflammation. This study contributes to the understanding EAAT and its relationship with intestinal mucositis, which can aid in the development of novel preventive strategies for cancer chemotherapy.

Translational PK-PD/TD modeling of antitumor effects and peripheral neuropathy in gemcitabine and nab-paclitaxel chemotherapy from xenograft mice to patients for optimal dose and schedule.

PURPOSE: Gemcitabine and nab-paclitaxel (GnP) treatment, the standard first-line chemotherapy for unresectable pancreatic cancer, often causes peripheral neuropathy (PN). To develop alternative dosing strategies to avoid severe PN, understanding the relationship between pharmacokinetics (PK) and pharmacodynamics/toxicodynamics (PD/TD) is necessary. We established a PK-PD/TD model of GnP treatment to develop an optimal dose schedule. METHODS: A mouse xenograft model of human pancreatic cancer was generated to measure drug concentrations in the plasma and tumor, antitumor effects, and PN after GnP treatment. The Simeoni tumor growth inhibition model with tumor concentrations and empirical indirect response models were used for the PD and TD models, respectively. Clinical outcomes were predicted with reported population estimates of PK parameters in cancer patients. RESULTS: The PK-PD/TD model simultaneously described the observed tumor volume and paw withdrawal frequency in the von Frey test. For the standard GnP regimen, the model predicted clinical overall response (75.1%), which was overestimated compared to that in a recent phase II study (42.1%) but lower than the observed disease control rate (96.5%). Model simulation showed that dose reduction to less than 40% GnP dose was not effective; a change of dose schedule from every week for 3 weeks to every 2 weeks was a more favorable approach than dose reduction to 60% every week. CONCLUSION: The PK-PD/TD model-based translational approach provides a guide for optimal dose determination to avoid severe PN while maintaining antitumor effects during GnP chemotherapy. Further research is needed to enhance its applicability and potential for combination chemotherapy regimens.

Cyclooxygenase (COX)-1 and COX-2 both play an important role in the protection of the duodenal mucosa in cats.

Although nonsteroidal anti-inflammatory drugs often cause ulcers in the duodenum in humans, the role of cyclooxygenase (COX) isoforms in the pathogenesis of duodenal ulcers has not been fully elucidated. We examined in cats the 1) ulcerogenic effects of selective COX-1 (SC-560, ketorolac) and COX-2 (celecoxib, meloxicam) inhibitors on the gastrointestinal mucosa, 2) effect of feeding and cimetidine on the expression of COX isoforms and prostaglandin E(2) (PGE(2)) level in the duodenum, and 3) localization of COX isoforms in the duodenum. COX inhibitors were administered after the morning meal in cats once daily for 3 days. Gastrointestinal lesions were examined on day 4. Localization and expression of COX isoforms (by immunohistochemistry, Western blot) and PGE(2) level (by enzyme immunoassay) were examined. Results were as follows. First, selective COX-1 or COX-2 inhibitors alone produced marked ulcers in the duodenum but did not cause obvious lesions in the small intestine. Coadministration of SC-560 and celecoxib produced marked lesions in the small intestine. Second, feeding increased both the expression of COX isoforms and PGE(2) level in the duodenum, and the effects were markedly inhibited by pretreatment with cimetidine. Third, COX-1 was localized in goblet and Brunner's gland cells, Meissner's and Auerbach's plexus, smooth muscle cells, and arterioles; and COX-2 was observed in capillaries, venules, and basal granulated cells. The expression of COX isoforms in the duodenum is up-regulated by feeding, and inhibition of either COX-1 or COX-2 causes ulcers in the duodenum, suggesting that both isoforms play an important role in the protection of the duodenal mucosa.

Effects of Helicobacter pylori infection on gastric parietal cells and E-cadherin in Mongolian gerbils.

Atrophic gastritis caused by infection with Helicobacter pylori is characterized by parietal cell loss, which is a main risk factor for gastric cancer. Parietal cells play a crucial role in the regulation of cell lineage maturation and proliferation in the gastric units. Among the classical cadherins, E-cadherin plays an important role not only in epithelial cell-cell connections, but also in the maintenance of epithelial polarity and gastric glandular architecture and regulation of cell proliferation. The aim of this study is to elucidate how parietal cells and E-cadherin are altered in gastritis with Helicobacter pylori infection. We studied the effects of Helicobacter pylori on gastric mucosal E-cadherin 2 weeks after inoculation and investigated the relationship between parietal cell loss and the amount of E-cadherin on parietal cells in Mongolian gerbils. The number of parietal cells and amount of staining of E-cadherin below the isthmus were investigated by immunohistochemistry. It was shown that a reduction in intercellular E-cadherin preceded the disappearance of parietal cells. The gastric glands where parietal cells were lost were replaced by mucus secreting cells without E-cadherin. These results suggest that Helicobacter pylori damaged E-cadherin on parietal cells and caused massive parietal cell loss, leading to the deregulation of gastric morphology.

The role of food for the formation and prevention of gastrointestinal lesions induced by aspirin in cats.

BACKGROUND/AIMS: The effects of feeding conditions (fasted or fed) and dietary fiber (DF) in the diet on gastrointestinal (GI) damage induced by aspirin (ASA) were examined in cats. METHODS: Plain ASA (P-ASA, 20 mg/kg) or one enteric-coated ASA tablet (EC-ASA, containing 100 mg ASA) was administered p.o. once daily for 3 or 7 days just after morning meal, 3 h after the evening meal, or in the morning without a morning meal (fasted). Several types of diet, dry food (DRY, DF: 2.8 %), canned food (CAN, DF: 0.4 %), and diets with added cellulose or pectin were provided twice daily. RESULTS: P-ASA or EC-ASA administered just after feeding of DRY caused marked lesions in the GI tract, although EC-ASA did not produce any lesions in the stomach. GI damage was markedly decreased when ASA was administered 3 h after the evening meal. The induction of lesions by EC-ASA was markedly decreased in cats that ate CAN, but lesions were induced in cats fed CAN with added cellulose (6 %). The addition of pectin (6 %) to the DRY markedly decreased the induction of lesions by EC-ASA. CONCLUSIONS: The results indicate that the induction of GI lesions by ASA was highly dependent on the feeding conditions and DF. To minimize the induction of GI damage, it would be better to take ASA 3 h after the evening meal, or after consuming diets that contain low amounts of insoluble DF and high amounts of soluble DF.

Mobilization of Circulating Tumor Cells after Short- and Long-Term FOLFIRINOX and GEM/nab-PTX Chemotherapy in Xenograft Mouse Models of Human Pancreatic Cancer.

Mobilization of CTCs after various types of therapy, such as radiation therapy, has been reported, but systematic study of CTCs after chemotherapy remained quite limited. In this study, we sequentially examined CTC numbers after single-dose and repetitive-dose chemotherapy, including FORFIRINOX (FFX) and Gemcitabine and nab-Paclitaxel (GnP) using two pancreatic cancer xenograft models. CTC was detected by the immunocytology-based microfluidic platform. We further examined the dynamic change in the histology of primary tumor tissues during chemotherapy. We confirmed a transient increase in CTCs 1-2 weeks after single-dose and repetitive-dose of FFX/GnP chemotherapy. Histological examination of the primary tumors revealed that the peak period of CTC at 1-2 weeks after chemotherapy corresponded to the maximal destructive phase consisting of cell cycle arrest, apoptosis of tumor cells, and blood vessel destruction without secondary reparative tissue reactions and regeneration of tumor cells. These findings indicate that mobilization of CTCs early after chemotherapy is mediated by the shedding of degenerated tumor cells into the disrupted blood vessels driven by the pure destructive histological changes in primary tumor tissues. These results suggest that sequential CTC monitoring during chemotherapy can be a useful liquid biopsy diagnostic tool to predict tumor chemosensitivity and resistance in preclinical and clinical settings.